Delineation of Borrelia burgdorferi motility and chemotaxis in the development of Lyme disease Lyme disease is caused by the spirochetal bacteria Borrelia burgdorferi (Bb), which is transmitted to humans by Ixodes ticks. The disease is categorized as an emerging infectious disease and is the most prevalent vector borne disease in the United States. Lyme disease has various clinical manifestations including erythema migrans rash, arthritis, carditis, and neurological symptoms. Though treatable with antibiotics, certain chronic cases are much more resistant, even to intravenous antibiotics. No vaccine is currently available, thus identification of virulence mechanisms that are important in causing Lyme disease is critical for developing effective prevention/treatment regimes. Bacterial motility and chemotaxis are central to the development of many infectious diseases. Although motility- and chemotaxis-associated genes constitute 6% of the Bb genome, a role for most of these genes in escaping immune clearance has not been described. Notably, while certain motility and chemotaxis mutants in many bacteria were shown to be less infectious or delayed in colonization processes, a similar mutant of Bb is non-infectious and cleared by mice within 24-48 hours, indicating motility and chemotaxis are vital for the enzootic life cycle of Bb. These findings agree with the proposed infection model, where tick-deposited bacteria quickly recognize and adapt to their new host, utilize their spirochetal motility to rapidly disseminate through dense skin tissues ahead of the cellular immune responses, and utilize chemotactic signals to reach immunoprivileged tissues where they can persist for long periods and evade the host antibody responses. Based on our preliminary results and published reports, we hypothesize that Bb motility and chemotaxis are essential for Bb virulence. The long term goal is to describe how Bb utilizes chemotaxis and motility to invade host tissues and evade immune clearance, allowing for disease development. Three specific aims are proposed to test this hypothesis: Aim 1 will generate targeted knockouts of 7 different motility and chemotaxis genes, and describe the motility/chemotaxis phenotype of these mutants in vitro. Aim 2 will delineate the relative infectivity of these mutants in vivo and reveal whether they can complete the natural "mouse-to-tick-to-mouse" infection cycle. Aim 3 will utilize multiphoton microscopy and novel intravital imaging techniques to directly visualize and describe how these mutant strains perform essential virulence properties within the skin tissues of living mice. Together, these studies will delineate the important functions and critical roles of each of these genes in Bb virulence and potentially identify targets for therapies to prevent and/or treat Lyme disease. PUBLIC HEALTH RELEVANCE: Lyme disease is the most common tick-borne disease in the United States and the rest of the Northern hemisphere, however no vaccine is currently available. The ability of Borrelia burgdorferi to use their novel spirochetal motility and chemotaxis properties to efficiently disseminate through dense host tissues and reach immunoprotective niches are believed to be critical to disease establishment. These studies will utilize a number of targeted mutants to directly delineate the importance of B. burgdorferi motility and chemotaxis genes for establishing infection in both tick and murine hosts, with the hope of identifying targets for curative therapies or vaccines.